Bacteria possess regulatory networks that sense environmental and metabolic conditions, and respond by generating and transducing signals that affect gene expression. Starvation and high cell density influence the production of virulence factors such as toxins, antibiotics, and degradative enzymes through regulatory networks. They also induce complex cell differentiation processes that give rise to resistant cell types or competent cells that can acquire exogenous DNA. In Bacillus subtilis, the srf operon resides within a regulatory network that governs processes induced by nutrient depletion and high cell density. srf encodes surfactin synthetase, an antibiotic biosynthesis operon, and ComS, a regulatory peptide that controls competence development. The major goal of the project is to understand how srf and comS are regulated and how ComS stimulates competence development. srf is under the control of two converging regulatory pathways. One mediates quorum-sensing control and involves the two-component regulators ComP and ComA; phorphorylated ComA activates srf transcription. The other pathway, involving the Phr extracellular peptide and the SpoOK oligopeptide permease, is activated by starvation and high cell density; the Phr peptide, imported via SpoOK, inhibits the Rap phosphatase that converts ComA to an inactive form, allowing interplay between the two pathways. Activation of phr expression requires the SigmaH form of RNA polymerase, the activity of which is induced by starvation and requires ClpX, an ATP-dependent chaperone. The role of ClpX in the activation of E-SigmaH will be determined by purification and reconstitution of RNA polymerase in vitro for transcription reactions containing purified ClpX proteins. Mutations which suppress the phenotype of a clpX mutant will be characterized to identify factors influencing ClpX-dependent activation of E-SigmaH. Other functions of ClpX in the activation of srf transcription will be identified by testing the effects of clpX comP and clpX spoOK double mutants on srf expression. ComS is required to release ComK, the transcriptional activator of competence gene expression, from the competence inhibitory proteins MecA and ClpC. The ComS-dependent release is thought to rescue ComK from regulated proteolysis. A collection of ComS point mutations will be analyzed to determine the function of ComS in the activation of competence gene expression. These studies will further understanding of the functional links between stress-induced proteins and the regulation of prokaryotic cellular differentiation.